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. 2006 May 16;103(20):7923-8.
doi: 10.1073/pnas.0602319103. Epub 2006 May 8.

Ripening in the tomato Green-ripe mutant is inhibited by ectopic expression of a protein that disrupts ethylene signaling

Cornelius S Barry  1 , James J Giovannoni
Affiliations

Ripening in the tomato Green-ripe mutant is inhibited by ectopic expression of a protein that disrupts ethylene signaling

Cornelius S Barry et al. Proc Natl Acad Sci U S A. .

Abstract

To achieve full ripening, climacteric fruits, such as tomato require synthesis, perception and signal transduction of the plant hormone ethylene. The nonripening phenotype of the dominant Green-ripe (Gr) and Never-ripe 2 (Nr-2) mutants of tomato is the result of reduced ethylene responsiveness in fruit tissues. In addition, a subset of ethylene responses associated with floral senescence, abscission, and root elongation are also impacted in mutant plants, but to a lesser extent. Using positional cloning, we have identified an identical 334-bp deletion in a gene of unknown biochemical function at the Gr/Nr-2 locus. Consistent with a dominant gain of function mutation, this deletion causes ectopic expression of Gr/Nr-2, which in turn leads to ripening inhibition. A CaMV35::GR transgene recreates the Gr/Nr-2 mutant phenotype but does not lead to a global reduction in ethylene responsiveness, suggesting tissue-specific modulation of ethylene responses in tomato. Gr/Nr-2 encodes an evolutionary conserved protein of unknown biochemical function that we associate here with ethylene signaling. Because Gr/Nr-2 has no sequence homology with the previously described Nr (Never-ripe) ethylene receptor of tomato we now refer to this gene only as GR. Identification of GR expands the current repertoire of ethylene signaling components in plants and provides a tool for further elucidation of ethylene response mechanisms and for controlling ethylene signal specificity in crop plants.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Structure of the Gr and Nr-2 loci. (A) Genetic and physical map of the Nr-2 locus based on 1,810 F2 plants. The number of recombinant individuals between adjacent markers is shown. BAC and cosmid clones are represented as horizontal bars, and approximate sizes are indicated (in kilobases). Four candidate genes at the Nr-2 locus are defined by their corresponding EST identifier. Arrows indicate the predicted direction of transcription. (B) Genomic structure corresponding to the EST clone cLPT12O9. Positions of primers used for RT-PCR (C1, C2, R) and amplification of genomic DNA (G1, G2) are shown. (C) RT-PCR amplification of cLPT12O9 from AC, Gr, and Nr-2 genotypes. SGN-U239539 was used as an equal loading control. (D) Identification of a deletion at the Gr and Nr-2 loci. Amplification of genomic DNA using the primers G1 and G2 is shown. The amplicon from control (AC) DNA is ≈1,400 bp. M refers to a DNA ladder.
Fig. 2.
Fig. 2.
CaMV35S::GR expression recreates the Gr phenotype. (A) Segregation of ripening inhibition in T1 progeny of four CaMV35S::GR independent transgenic lines (4, 6, 7, and 8). Normal ripening fruit (−) have segregated out the transgene, whereas nonripening fruit (+) have retained the transgene. Wild-type (AC) and Gr fruit of identical age are shown for comparison. (B) GR expression in fruit samples shown in A. Total RNA (20 μg) extracted from normal ripening fruit was hybridized to a 32P-labeled GR probe. The filter was stripped and reprobed with an 18S rRNA probe. (C) Petal retention on developing fruits of multiple CaMV35S::GR transgenic lines. (D) Frequency of ethylene-induced floral abscission. Floral abscission was monitored in wild type (AC), Gr, and two homozygous CaMV35S::GR lines (4-20 and 6-14) 72 h after ethylene treatment. The mean of three independent experiments derived from at least 134 flowers is presented. Vertical bars represent SE.
Fig. 3.
Fig. 3.
Seedling triple response phenotype in CaMV35S::GR transgenic lines. Seeds of five different genotypes: wild type (AC), Nr, Gr, and CaMV35S::GR lines 4-20 and 6-14 were surface sterilized and sown on 0.8% water agar containing ACC at the indicated concentrations. Hypocotyl (A) and root (B) lengths were determined 8 days after sowing. Each data point is the mean of at least 31 seedlings. Vertical bars represent SE. (C) GR expression in hypocotyls of etiolated seedlings. Total RNA (20 μg) extracted from the genotypes described in A was hybridized to a 32P-labeled GR probe. The filter was stripped and reprobed with an 18S rRNA probe.
Fig. 4.
Fig. 4.
Phylogenetic analysis of GR related proteins. A nonrooted phylogenetic tree was generated by using the phylip 3.5c suite of programs (http://evolution.genetics.washington.edu/phylip.html) with the C. elegans protein as the out-group. The single most parsimonious tree obtained in a heuristic search following 100 random sequence addition replicates is shown. Bootstrap percentage supports are indicated. Sequence identifiers are available in Supporting Text.
Fig. 5.
Fig. 5.
GR expression is associated with seed development. (A) GR expression during fruit ripening in wild type (AC) and Gr. Stages 1–5 are defined in ref. . (B) GR expression in mature green fruit with (+) or without (−) ethylene treatment. (C) GR expression during tomato seed development. RNA was extracted from seeds of wild-type tomato fruit at the immature green (2), mature green (3), and red-ripe (4) stages of development. RNA extracted from Gr mature green fruit pericarp tissue was included as a positive control (1). (D) GR expression in wild-type and mutant seeds. Seeds were extracted from immature wild-type (1) and Gr (2) fruit. All blots contained 15 μg of total RNA and were hybridized to a 32P-labeled GR probe. Images of the rRNA are shown as a guide to equal loading.
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